| Traditional solid fermentation is a food processing method with a variety of complex microorganisms involved inside, which has been considered as one of the most ancient and economical means for food production and storage. The method can notably increase the nutritional value, organoleptic properties and functional characteristics of raw food materials,thus making researches on microbial diversity(especially on the diversity of uncultured microorganisms) and microbial functional characteristics during the solid-state fermentation process become more and more important. As a critical process for the production of Chinese traditional fermented vinegar, solid-state acetic acid fermentation has endowed vinegar products with typical flavors and functional characteristics via the transformation of grain raw materials into diverse flavor substances and functional substances with the participation of various microorganisms, which makes itself to be an important seasoning and medicine in Chinese people's daily life. Recently, a lot of microbiology researches have revealed the microbial community diversity as well as its formation rules in the traditional brewing process of fermented vinegar. However, both microorganism interactions in the community and mutual relationship among the main metabolites are still unknown. This paper established an integrated database for food microorganisms(FoodMicrobes) incorporating the management,inquiry and analysis of metagenome data as a whole through data information integration of food microorganisms across different omics levels by applying the method of semantic web data division. Zhenjiang aromatic vinegar was chosen as the research object and such molecular ecology methods as metagenome sequencing were used to build metabolic network of main flavor substances in the vinegar microbial communities, with the microbial distribution and functional differences from different metabolic pathways being analyzed.Based on the information acquired from metagenome sequencing, the synthetic route of diacetyl/acetoin in the vinegar microbial community was selected as research object to separate and obtain microbial strains capable of synthesizing acetoin, with functions of the microbial strains under both ectopic and in-situ condition being verified. The major results of the research are as follows:Systematic researches on the ontology of associated data in microbial field and design approach of namespace were done. The corresponding ontology and namespaces were established for main data types which were relevant to food microorganisms. FoodMicrobes database was established with the associated data and data warehouse created through applying the Resource Description Framework(RDF). It not only achieved the data integration of food microorganisms across various levels, but also systematically combed therelations ranging from the mutual relationship among macroscopic data including microorganism strains and types to that among microcosmic data concerning genomes,proteins, enzymes and metabolic pathways, eventually forming a tightly connected system.Tools and algorithms for metagenome data analysis including BLAST, genome visualization tool, gene functional annotation tool, metabolic pathway analysis tool and metabolic pathways visualization tool, were integrated to build a data platform incorporating the management, inquiry and analysis of metagenome data as a whole, which provided a necessary tool for data analysis of microbial community during the brewing process.Metagenome sequencing technology was applied to detect and analyze the sequence of metagenome in the Zhenjiang fragrant vinegar microbial community during the brewing process. The 54340 contig was produced by Metavelvet and genes within the contig were scanned with FragGeneScan, with 86201 protein-coding regions being produced. The analysis result shown that the relative abundances of bacteria, eukaryons and archaebacterias were91.75%, 0.19% and 8.06% respectively in the vinegar microbial community. As for the Phylum level, firmicutes(48.16%) and proteobacteria(6.83%) were regarded as the major microbial communities of Zhenjiang fragrant vinegar. According to the gene information obtained from KEGG pathway annotation, 2636 genes belong to metabolic process, together with 944 genes involved in genetic information processing, 538 genes related to cellular processes, 527 genes involved in environmental information processing, 658 genes relevant to human diseases and 378 genes belonging to organism system.Microbial metabolic network concerning the main flavor substances of Zhenjiang fragrant vinegar was built in accordance with information acquired from functional annotation of metagenome. Species and function associations related to metagenome sequencing were built by adopting effective components and metagenomie composition vector(MetaCV) to obtain the distributional differences of microbial species and their functions in different metabolic pathways. In terms of the metabolic network of main flavor substances in the Zhenjiang fragrant vinegar, acetoin, diacetyl, isoamylol, tetramethylpyrazine and butanediol were the main volatile ingredients in the Zhenjiang fragrant vinegar and its culture medium,with the leucine and valine being the main amino acid, acetoin and diacetyl being the precursor to ligustrazine through Maillard reaction and ligustrazine being the main flavor and active substance. Therefore, it was important to participate in both acetoin-diacetyl-butanediol group and 3-methyl butanol-3-methyl butyric acid-valine-leucine group for the formation of special vinegar flavor. On account of the differences of these two microbial functional groups,a lot of microorganisms would participate in the 3-methyl butanol-3-methyl butyric acid-valine-leucine group, while those involved in the acetoin-diacetyl-butanediol group were mainly actinomycetes, lactobacillus, hymenoscyphus and oboediens.Acetoin/diacetyl metabolic pathway was chosen as the research object and the production potential of special flavor from complex microflora were accurately evaluated based on omics-derived data. Furthermore, the usable information of deep sequencing in vinegar microbial community was utilized to explore the functional microbial strains,including acetobacter pasteurianus, lactobacillus buchneri, lactobacillus fermenti,lactobacillus brevis strains, capable of synthesizing acetoin via directional separation during brewing process of Zhenjiang fragrant vinegar. In addition, the metabolism of functional microbial strains were verified experimentally at the shake flask level and in-situ level. It was testified that the addition of acetobacter pasteurianus G3-2, lactobacillus brevis, lactobacillus fermenti M10-3 and lactobacillus buchneri F2-5 to microbial community during vinegar brewing could regulate its metabolism and enhance the effective synthesis of acetoin. |
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